Program Director/Principal Investigator (Last, First, Middle): GUAN, JIANJUN Project Summary Critical limb ischemia (CLI) is a severe peripheral artery disease with high rates of limb loss and mortality. It is featured by low blood perfusion, extensive tissue ischemia, and degenerated skeletal muscle. Quick vascularization to restore blood perfusion, and fast muscle regeneration to restore normal function, represent the optimal goals for CLI treatment. Currently there is no efficient treatment available, although stem cell therapy is one of the most promising strategies. Most of stem cell types promote vascularization and muscle regeneration mainly by paracrine effects while some may also differentiate into endothelial and skeletal muscle cells. However, current stem cell therapy experiences low efficacy largely due to inferior cell survival and paracrine effects under the extremely low oxygen condition (<1%) of ischemic limbs. In this project, we propose a new cell delivery system that continuously releases appropriate concentration of O2 to simultaneously improve stem cell survival and paracrine effects, resulting in quick vascularization and muscle regeneration. Paracrine effects concurrently provide multiple growth factors critical for vascularization and muscle regeneration, which cannot be readily achieved by growth factor therapy. In our preliminary studies, we have created a hydrogel-based cell delivery system that releases O2. When tested using bone marrow-derived mesenchymal stem cells (MSCs), the released O2 increased cell survival under ischemic conditions in vitro without increasing reactive oxygen species (ROS) content. It also upregulated MSC paracrine effects especially in terms of secreting proangiogenic/promyogenic growth factors like PDGF and IGF-1. After implanting into ischemic limbs, the O2 releasing cell delivery system not only augmented MSC survival, but also fully restored blood perfusion and muscle contractility in 4 weeks. The contribution of MSCs to vascularization is mainly from paracrine effects as only a low percentage of cells were differentiated into endothelial cells. Meanwhile, both MSC paracrine effects and myogenic differentiation contributed to muscle regeneration. These preliminary data suggest that increasing both MSC survival and paracrine effects can significantly enhance vascularization and muscle regeneration in ischemic limbs. Yet, cell survival and paracrine effects do not always increase concurrently. Based on our preliminary studies and above discussion, we hypothesize that stem cell delivery systems with optimal O2 release profiles that simultaneously increase MSC survival and paracrine effects, will significantly accelerate vascularization and muscle regeneration in ischemic limbs. Aim #1 will test the hypothesis that optimal O2 release profiles will promote MSC survival and paracrine effects under ischemic conditions. Aim #2 will test efficacy of the created cell delivery systems using a model of hindlimb ischemia. This project is innovative because it develops a safe and long-term O2 release system to establish the role, mechanism, and efficacy of controlled O2 release in augmenting both stem cell survival and paracrine effects in ischemic tissues for accelerated regeneration. The proposed stem cell delivery system is also translational. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page